Bottom Line:
Our characterization of Nse5 establishes a previously unidentified relationship between the Smc5/6 complex and factors of the SUMO pathway.Nse5 physically associates with the E2 conjugating enzyme, Ubc9, where contacts are stabilized by non-covalent interactions with SUMO.Overall, given the extensive connection between Nse5 and components of the SUMO pathway, we speculate that one function of the Smc5/6 complex might be as a scaffold center to enable sumoylation events in budding yeast.

BIO018440F2: Nse5 interactions with the PIAS family of E3 SUMO ligases, Siz1 and Siz2, and the E2-conjugating enzyme, Ubc9, is mediated by SUMO. (A) Two-hybrid analysis to determine interactions between bait: Nse5-LexA (J-038) vector and prey vectors with the four budding yeast SUMO ligases: either empty vector (J-1493), Siz1- (J-096), Siz2- (J-047), Mms21- (J-050) and Cst9-AD (J-095). Error bar represent the standard deviation from the mean of n=3 biological experiments performed in technical triplicate. *P<0.01; statistically different interaction between Nse5 and the E3 ligases than with the interaction between Nse5 and the vector alone (background), from a two-tailed t-test in A. (B) Two-hybrid analysis as described in A comparing wild-type (JC470) and smt3-331 (JC2758) mutant cells. *P<0.01; statistically different interaction between Nse5 with Siz1 or Siz2 in cells harboring the smt3-11 mutation compared to the same interactions measured in wild-type cells, from a two-tailed t-test. Representative western blots in Fig. S2A show the expression from the two-hybrid constructs. (C) Using the His-Smt3 Ni-NTA purification system, sumoylated Smc5 was purified from cells endogenously Myc-tagged Smc5 with un-tagged Smt3 wild-type (JC720), or His8-tagged Smt3 wild-type (JC1157), nse5-ts1 (JC1156), mms21-11 (JC1155), siz1Δ (JC2527), and siz2Δ (JC1964) mutant cells following 0.3% MMS treatment at 25°C. (D) Two-hybrid analysis was performed to determine interactions between bait: Nse5-LexA (J-038) and prey vectors: either empty pJG4-6 vector (J-1493) or Ubc9-AD (J-042) in WT (JC470) or smt3-331 (JC2758) cells. *P<0.01; statistically different interaction between Nse5 with Ubc9 in cells harboring the smt3-11 mutation compared to the same interactions measured in wild-type cells, from a two-tailed t-test. All interaction between Nse5 and Ubc9 were statistically different than the interaction between Nse5 and the vector alone (background) in both wild-type and smt3-11 cells with P<0.01. (E) Two-hybrid analysis showed no statistically significant differences in interactions between bait: Nse5-LexA (J-038) with the prey constructs: Ubc9 (J-042, wild-type), Ubc9-C93A (J-108, catalytic site mutant), Ubc9-K153R (J-101, SUMO-acceptor site mutant), Ubc9-C93A/K153R (J-110, SUMO-acceptor site and catalytic site double mutant). All interactions were significantly different than background levels with Nse5-LexA (J-038) and empty vector pJG4-6 (J-1493). Western blot analysis in Fig. S2B shows the expression from two-hybrid constructs at comparable levels.

Mentions:
Nse5 was previously shown to interact with SUMO (Hazbun et al., 2003; Bustard et al., 2012) and from the same high throughput Y2H screen (Hazbun et al., 2003), Nse5 was also reported to potentially interact with the E3 SUMO ligase, Siz2, and the E2 conjugating enzyme Ubc9. To verify these observations and expand these analyses of Nse5, Y2H was performed between Nse5 and the four known E3 SUMO ligases in budding yeast. Statistically significant interactions were observed between Nse5 with all E3 ligases except Cts9, the meiosis-specific E3 (Fig. 2A) (Cheng et al., 2006).Fig. 2.

BIO018440F2: Nse5 interactions with the PIAS family of E3 SUMO ligases, Siz1 and Siz2, and the E2-conjugating enzyme, Ubc9, is mediated by SUMO. (A) Two-hybrid analysis to determine interactions between bait: Nse5-LexA (J-038) vector and prey vectors with the four budding yeast SUMO ligases: either empty vector (J-1493), Siz1- (J-096), Siz2- (J-047), Mms21- (J-050) and Cst9-AD (J-095). Error bar represent the standard deviation from the mean of n=3 biological experiments performed in technical triplicate. *P<0.01; statistically different interaction between Nse5 and the E3 ligases than with the interaction between Nse5 and the vector alone (background), from a two-tailed t-test in A. (B) Two-hybrid analysis as described in A comparing wild-type (JC470) and smt3-331 (JC2758) mutant cells. *P<0.01; statistically different interaction between Nse5 with Siz1 or Siz2 in cells harboring the smt3-11 mutation compared to the same interactions measured in wild-type cells, from a two-tailed t-test. Representative western blots in Fig. S2A show the expression from the two-hybrid constructs. (C) Using the His-Smt3 Ni-NTA purification system, sumoylated Smc5 was purified from cells endogenously Myc-tagged Smc5 with un-tagged Smt3 wild-type (JC720), or His8-tagged Smt3 wild-type (JC1157), nse5-ts1 (JC1156), mms21-11 (JC1155), siz1Δ (JC2527), and siz2Δ (JC1964) mutant cells following 0.3% MMS treatment at 25°C. (D) Two-hybrid analysis was performed to determine interactions between bait: Nse5-LexA (J-038) and prey vectors: either empty pJG4-6 vector (J-1493) or Ubc9-AD (J-042) in WT (JC470) or smt3-331 (JC2758) cells. *P<0.01; statistically different interaction between Nse5 with Ubc9 in cells harboring the smt3-11 mutation compared to the same interactions measured in wild-type cells, from a two-tailed t-test. All interaction between Nse5 and Ubc9 were statistically different than the interaction between Nse5 and the vector alone (background) in both wild-type and smt3-11 cells with P<0.01. (E) Two-hybrid analysis showed no statistically significant differences in interactions between bait: Nse5-LexA (J-038) with the prey constructs: Ubc9 (J-042, wild-type), Ubc9-C93A (J-108, catalytic site mutant), Ubc9-K153R (J-101, SUMO-acceptor site mutant), Ubc9-C93A/K153R (J-110, SUMO-acceptor site and catalytic site double mutant). All interactions were significantly different than background levels with Nse5-LexA (J-038) and empty vector pJG4-6 (J-1493). Western blot analysis in Fig. S2B shows the expression from two-hybrid constructs at comparable levels.

Mentions:
Nse5 was previously shown to interact with SUMO (Hazbun et al., 2003; Bustard et al., 2012) and from the same high throughput Y2H screen (Hazbun et al., 2003), Nse5 was also reported to potentially interact with the E3 SUMO ligase, Siz2, and the E2 conjugating enzyme Ubc9. To verify these observations and expand these analyses of Nse5, Y2H was performed between Nse5 and the four known E3 SUMO ligases in budding yeast. Statistically significant interactions were observed between Nse5 with all E3 ligases except Cts9, the meiosis-specific E3 (Fig. 2A) (Cheng et al., 2006).Fig. 2.

Bottom Line:
Our characterization of Nse5 establishes a previously unidentified relationship between the Smc5/6 complex and factors of the SUMO pathway.Nse5 physically associates with the E2 conjugating enzyme, Ubc9, where contacts are stabilized by non-covalent interactions with SUMO.Overall, given the extensive connection between Nse5 and components of the SUMO pathway, we speculate that one function of the Smc5/6 complex might be as a scaffold center to enable sumoylation events in budding yeast.